<br>FREQ-Seq2：一种精确高通量组合定量等位基因频率的方法,G3: Genes, Genomes, Genetics

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FREQ-Seq2：一种精确高通量组合定量等位基因频率的方法
G3: Genes, Genomes, Genetics ( IF 2.1 ) Pub Date : 2023-07-26 , DOI: 10.1093/g3journal/jkad162
Roy Zhao ₁ , Tamas Lukacsovich ₂ , Rebecca Gaut ₃ , J J Emerson _{1,

3}

Affiliation

准确确定等位基因频率对于遗传学中的各种问题至关重要，例如开发群体遗传模型、从全基因组关联研究中进行推论、确定疾病的遗传风险以及其他科学和医学应用。此外，了解种群中等位基因频率如何随时间变化对于确定其进化动态至关重要。我们提出了一种精确、高效且经济的方法 (FREQ-Seq2)，用于量化混合群体样本中感兴趣位点的不同等位基因的相对频率。通过创造性地使用配对条形码序列，我们将原始 FREQ-Seq 方法的吞吐量从 48 个样本指数级增加到 2,304 个样本。 FREQ-Seq2 可以靶向特定的感兴趣基因组区域，使用通用条形码接头进行扩增以生成 Illumina 测序文库。我们的增强方法与用于分析测序文库的开源软件一起作为套件提供，能够检测和消除在原始 FREQ-Seq 方法以及其他传统等位基因频率定量方法中无法检测到的错误。最后，我们使用一组高度多重的对照样本以及大肠杆菌的竞争进化实验验证了基于 NGS 的方法的性能，并将后者与手动菌落计数得出的估计值进行比较。我们的分析表明，FREQ-Seq2 灵活、廉价，并且能够生成大量具有低误差、低噪声和理想统计特性的数据。总之，FREQ-Seq2 是一种用于量化等位基因频率的强大方法，为分析混合群体提供了一种通用方法。

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FREQ-Seq2: a method for precise high-throughput combinatorial quantification of allele frequencies

The accurate determination of allele frequencies is crucially important across a wide range of problems in genetics, such as developing population genetic models, making inferences from genome-wide association studies, determining genetic risk for diseases, as well as other scientific and medical applications. Furthermore, understanding how allele frequencies change over time in populations is central to ascertaining their evolutionary dynamics. We present a precise, efficient, and economical method (FREQ-Seq2) for quantifying the relative frequencies of different alleles at loci of interest in mixed population samples. Through the creative use of paired barcode sequences, we exponentially increased the throughput of the original FREQ-Seq method from 48 to 2,304 samples. FREQ-Seq2 can be targeted to specific genomic regions of interest, which are amplified using universal barcoded adapters to generate Illumina sequencing libraries. Our enhanced method, available as a kit along with open-source software for analyzing sequenced libraries, enables detection and removal of errors that are undetectable in the original FREQ-Seq method as well as other conventional methods for allele frequency quantification. Finally, we validated the performance of our NGS-based approach with a highly multiplexed set of control samples as well as a competitive evolution experiment in E. coli, and compare the latter to estimates derived from manual colony counting. Our analyses demonstrate that FREQ-Seq2 is flexible, inexpensive, and produces large amounts of data with low error, low noise, and desirable statistical properties. In summary, FREQ-Seq2 is a powerful method for quantifying allele frequency that provides a versatile approach for profiling mixed populations.

更新日期：2023-07-26

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